Novel adult cortical neuron processing and screening method illustrates sex- and age-dependent effects of pharmaceutical compounds

Neurodegenerative diseases and neurotraumatic injuries are typically age-associated disorders that can reduce neuron survival, neurite outgrowth, and synaptic plasticity leading to loss of cognitive capacity, executive function, and motor control. In pursuit of reducing the loss of said neurological functions, novel compounds are sought that promote neuron viability, neuritogenesis, and/or synaptic plasticity. Current high content in vitro screenings typically use cells that are iPSC-derived, embryonic, or originate from post-natal tissues; however, most patients suffering from neurodegenerative diseases and neurotrauma are of middle-age and older. The chasm in maturity between the neurons used in drug screens and those in a target population is a barrier for translational success of in vitro results. It has been historically challenging to culture adult neurons let alone conduct screenings; therefore, age-appropriate drug screenings have previously not been plausible. We have modified Miltenyi’s protocol to increase neuronal yield, neuron purity, and neural viability at a reduced cost to expand our capacity to screen compounds directly in primary adult neurons. To our knowledge, we developed the first morphology-based screening system using adult cortical neurons and the first to incorporate age and sex as biological variables in a screen using adult cortical neurons. By using primary adult cortical neurons from mice that were 4 to 48 weeks old for screening pharmaceutical agents, we have demonstrated age- and sex-dependent effects on neuritogenesis and neuron survival in vitro. Utilizing age- and sex-appropriate in vitro models to find novel compounds increasing neuron survival and neurite outgrowth, made possible by our modified adult neuron processing method, will greatly increase the relevance of in vitro screening for finding neuroprotective compounds.

www.nature.com/scientificreports/ we identified compounds with age-and sex-dependent effects on neuron survival and neurite outgrowth. This clearly illustrates the need to perform future screens in different age and sex groups (1) to determine the specific demographic the compound will have most clinical effect in; (2) to theoretically prevent the premature dismissal of compounds with no effect in iPSC/embryonic screens yet would be advantageous to adults; and (3) to reduce the false positivity rate from iPSC/embryonic screens by efficiently vetting compounds that do not show benefit to adult neurons and therefore will likely not be efficient when testing in preclinical settings.

Results
The original protocol from Miltenyi has been extensively modified and optimized to fit various drug screening criteria, by processing larger amount of tissue, expediting the process, reducing costs, and providing researchers with a new tool that can potentially provide more clinically relevant data than previous conventional screening methods. Importantly, we demonstrated the utility of this system by illustrating the sex and age-dependent effects of 2 compounds.
Optimization of surface coating and media supplementation. We first determined if laminin coating in addition to the PDL would improve neurite outgrowth and number of valid neurons and/or mitigate the need for extra supplementation of media with B27 + . Primary cortical neurons isolated from young adult male mice, using the original Miltenyi protocol 59 , were cultured at 10,000 cells/well for 2DIV on PDL or PDL/laminin coated wells with or without B27 +. The B27 + supplementation significantly increased the average neurite length (Fig. 1A , P < 0.05), total neurite outgrowth (total neurite outgrowth, P < 0.0001), and number of valid neurons ( Fig. 1C , P < 0.0001). The presence of laminin did not improve neurite growth or the number of valid neurons, nor can it replace or enhance media supplementation. Plating neurons without PDL coating resulted in few www.nature.com/scientificreports/ neurons adhering to the surface without substantial neurite growth (data not included). Media supplementation and surface coating is required, especially to increase the number of valid adult cortical neurons. Only PDL coating was used for subsequent experiments and all experimentation was done in standard size 384-well plates.
Determine the best digestion enzyme. Next, we determined the effectiveness of different digestion enzymes and concentrations to use to isolate cortical neurons and their efficacy to maintain their viability. Identical dissection method, dissociation method and temperature, and volumes of digestive enzymes were used herein. After the respective digestive enzymes were used in replacement of 0.3 mg/mL papain ("Methods" section) to isolate cortical neurons from young adult male mice (1 mouse cortex per condition), the neurons were evenly distributed between 6 wells and cultured for 2DIV to measure how many cortical neurons can be extracted using each digestive enzyme and their ability to maintain neuron viability ( Fig. 1D-H). Overall, papain presented beneficial effects on cell survival and neurites outgrowth compared to the MACS ® P&A digestive enzyme. 0.3-1.0 mg/mL papain resulted in increased total neurite outgrowth (P < 0.0001) and number of valid neurons (P < 0.0001) when compared to MACS ® P&A. 0.1 mg/mL papain had significantly higher total neurite outgrowth and number of valid neurons compared to MACS ® P&A (P < 0.0001), yet significantly lower values relative to higher papain concentrations (P < 0.0001). This suggests digesting with papain has a dosedependent effect that plateaus at ≤ 0.3 mg/mL. Notably, 0.1 mg/mL papain had higher average neurite length compared to 0.5 mg/mL papain (P < 0.01) and MACS ® P&A (P < 0.001), although still had similar total neurite outgrowth compared to 0.5 mg/mL papain. There were no significant differences between papain concentrations of 0.3-1.0 mg/mL for any analysis, and therefore, the concentration of 0.3 mg/mL, the lowest effective papain concentration, was used for the remainder of the experiments to reduce cost and potential off-target effects induced by higher concentrations. Accutase ® and 0.25% Trypsin were also tested and yielded poorer outcomes compared to MACS ® P&A (data not included).
Determine the best dissociation method, timing, and temperature. Here, we determined the effectiveness of different digestion methods, the incubation timing of those methods, and the incubation temperatures on the dissociation of cortical tissue from young adult male mice. After the cortical neurons preparation with each respective protocol, the cells are evenly dispersed between 6 wells to analyze both the yield and viability of the neurons. We compared the standard protocol ABDK-30 min to ABDK-20 or 10 min (all in the Octo Dissociator with heaters at 37 °C) and to a revolving apparatus at different times and temperatures (Fig. 2). Neurons dissociated using the Octo Dissociator had longer average neurite lengths and total neurite outgrowth in comparison to neurons incubated in a revolving apparatus. The incubation time had no effect on neuron morphology when using Octo Dissociator, yet, when using a rotating apparatus, a reduction in time lead to reduced total neurite growth (P < 0.0001). The number of valid neurons was affected by method, timing, and temperature. Using the Octo Dissociator increased the number of valid neurons by approximately fourfold regardless of the incubation time relative to using a revolving apparatus at 37 °C (P < 0.0001). In each dissociation method, there was approximately a twofold increase in the number of valid neurons for every 10 min increase in incubation time (P < 0.0001). Furthermore, reducing the temperature from 37 to 25 °C during the 20 min incubation in a revolving apparatus reduced the number of valid neurons without impacting neuron morphology (P < 0.0001, Fig. 2C). The gentleMACS Program 37C_ABDK_01 protocol was used in the following experiments.
The effects of cell plating density. The reaction of neurons to compounds may be dependent on their plating density. Therefore, we assessed the effects of cell plating density of cortical neurons (from young adult male mice) on the average neurite length, total neurite outgrowth, and number of valid neurons per well (Fig. 3). (S)-H-1152 is a selective and potent rho-associated kinase (ROCK) inhibitor that attenuates KCl-induced con- Figure 2. The effects of different dissociation protocols, timing, and temperature. Histograms of the (A) average neurite length, (B) total neurite outgrowth, and (C) number of valid neurons expressed as percent change relative to the standard gentleMACS Program 37C_ABDK_01 protocol (ABDK (30 min)). Data analyzed using one-way ANOVA with Dunnett's post-hoc test comparing the mean of each condition to the mean of ABDK (30 min). In parentheses represents the length of the protocol whether being ran on the gentleMACS™ Octo Dissociator with Heaters (ABDK) or in a revolving apparatus incubated at 37 °C or 25 °C. Primary cortical neurons isolated from young adult male mice and cultured for 2DIV. 6 wells per condition (n = 6). *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P < 0.0001). Graphs show mean and SEM. www.nature.com/scientificreports/ tractions of femoral arteries 60 and augments neurite outgrowth in dorsal root ganglion cells isolated from 1-day old rats that are cocultured with Schwan cells 61,62 . No significant correlation was found in the linear regression between plating density and the average neurite outgrowth for both 0.05% DMSO (dimethyl sulfoxide, Vehicle) and 5 µM (S)-H-1152 treated groups. There was a significant positive correlation between both total neurite outgrowth (P values: Vehicle < 0.001, (S)-H-1152 < 0.0001) and number of valid neurons per well (P values: Vehicle < 0.001, (S)-H-1152 < 0.0001) and the cell plating density for both treatment groups. Only the slope of the total neurite outgrowth linear regression line differed significantly between treatment groups, (S)-H-1152 induced a steeper increase in total neurite outgrowth and therefore more responsive to increased plating density (P < 0.05). (S)-H-1152 significantly increased the average neurite length (P < 0.05) and number of valid neurons (P < 0.01) compared to DMSO control only when 10,000 cells were plated per well. Therefore, plating density can determine which compounds are deemed positive hits and must be taken into consideration during screenings. The number of valid neurons for both Vehicle (R 2 = 0.9911) and (S)-H-1152 (R 2 = 0.9923) increases exponentially. Therefore, there may be extrinsic factors related to plating density that augment the neurite formation and/ or survival of neurons. To minimize the impact of these factors when screening for new compounds, which may hinder the effect of the tested drugs, the concentration of 10,000 neurons per well appears the best choice and was used in the rest of the experiments.
The efficacy of various neuronal supplements. To improve the survival of neurons and create an environment that resembles in vivo conditions, neuron supplements are added to media to study the synaptic function, neurite growth, and survival of primary neurons in vitro in a chemically defined manner without the use of serum [63][64][65][66] . Here, we determined the efficacy of NeuroBrew ® -21, B27 + , and NeuroCult™ SM1 as serum-free neuronal supplements to support neuron survival and neurite outgrowth of cortical neurons from young adult male mice (Fig. 4). The use of B27 + resulted in an increase in the average neurite length (P < 0.0001), total neurite outgrowth (P < 0.001), and number of valid neurons (P < 0.05) relative to all other cohorts. Therefore, B27 + will be used in future screenings to better nurture the adult neurons. The potential issue in using B27 + is the fact that it may mask the true effects of compounds due to its increased potency to augment survival and neurite growth compared to other supplements, although, compound screenings cannot be done in neurons that are not viable and reactive to drug treatment. On another note, there may be ingredients in the supplement that counteract www.nature.com/scientificreports/ potentially beneficial compounds or conceal their effects, therefore, we aim to minimize the need for supplementation and only use the recommended concentration of 1×.
Culture purity assessed with RNA expression analysis. The differences between the original and modified neuron isolation, processing, and culturing protocols are evident in the analysis for neurite outgrowth and valid neurons. These modifications have allowed for the culturing and screening of older adult cortical neurons. We performed RNA expression analysis on cortical neurons extracted from young adult male mice using RT-qPCR to determine how these modifications have affected the culture purity, by assessing RNA yield, neuron specific yield, and purity (Fig. 4). The RT-qPCR data indicates that the modified protocol resulted in significantly more RNA being isolated from each cortex (P < 0.0001), greater NeuN expression from the isolated cells (P < 0.01), and increased NeuN expression relative to GFAP and GLAST (P < 0.05). The in vitro cultures were suggestive of the modified protocol containing more cells; this data confirms that not only are more cells isolated, but a larger percentage of those cells are neurons. The modified protocol increased RNA yield by only 66% (Fig. 4A) yet increased the NeuN expression by 112% (Fig. 4B), suggesting an unproportionally larger increase in neurons relative to other neural cells. This was confirmed by the significant increase in the relative NeuN expression in the modified protocol compared to the combination of GLAST and GFAP expression (Fig. 4C). To confirm with immunocytochemistry, we stained the cell cultures at 2DIV with GFAP and Iba1 and found none of the cells to be positive for either marker in either method (data not shown).
The effects of the vehicle DMSO. Due to the ability of DMSO to dissolve hydrophobic compounds 67 , it is commonly used as a vehicle in high content screenings 68,69 . To determine the tolerance of adult neurons to DMSO, we plated primary cortical neurons from young adult male mice in presence of different concentrations of DMSO using the modified protocol (Fig. 5). As expected, there was significant negative correlation www.nature.com/scientificreports/ between average neurite length (P < 0.05), total neurite outgrowth (P < 0.01), and number of valid neurons per well (P < 0.01) and the percentage of the media containing DMSO (% DMSO).
Sex and age-dependent effects of RO48 and culturing methods. We analyzed the effects of our isolation method, media, culturing protocol, and Vehicle against the young adult and middle-aged female cohorts. The middle-aged female cohort had a small but significant increase in average neurite length and significant decrease in total neurite outgrowth compared to the younger female cohort (Fig. 6A,B). The middleaged female cohort has a non-significant downward trend in number of valid neurons compared to the younger  Data analyzed using student's T-test comparing the means of each cohort denoted by *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P < 0.0001). Linear trends of the (D) average neurite length, (E) total neurite outgrowth, and (F) number of valid neurons of primary cortical neurons isolated from young adult male, young adult female, and middle-aged female mice cultured in various concentrations of RO48 for 2DIV. The X-axis denotes the concentration (nM) of RO48 in the media (D-F). The values are expressed as percent change relative to the Vehicle treatment group of each cohort. Representative × 20 magnification images of (G) Vehicle and (H) 3400 nM RO48 treated primary cortical neurons isolated from middle-aged female mice and cultured for 2DIV; stained with TUBB3 (Green) and DAPI (Blue). Simple liner regression was conducted to determine goodness of fit and if the slope differs significantly from 0. Linear regression t-test was used to compare the slope of the regression lines. Two-way ANOVA with Šídák's multiple comparisons test was used to compare the means of neurons treated with 3400 nM RO48 between cohorts denoted by # (P < 0.05), ## P < 0.01), ### (P < 0.001), #### (P < 0.0001) and to compare the means of neurons treated with various concentrations of RO48 to the Vehicle treatment group of the respective cohort denoted by *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P < 0.0001). All cohorts have equal parts Vehicle in media (0.05% DMSO). 3 wells per condition (n = 3). Graphs show mean and SEM. Scale Bar = 50 µm. www.nature.com/scientificreports/ female cohort (Fig. 6C). RO48 activates mammalian target of rapamycin complex (mTORC)1/2 and phosphatidylinositol-3-kinase (PI3K) and decreases the phosphorylation of S6-926 70 . RO48 has been shown to promote axon regeneration in vitro and augment axon growth and functional locomotor recovery in a rodent spinal cord injury model 71 . Therefore, RO48 is expected to provide neuroprotective qualities and/or augment neurite growth in our screen. There was a significant positive correlation between the RO48 concentration in the media and the total neurite outgrowth (P < 0.01) and number of valid neurons (P < 0.05) in the young adult female cohort only ( Fig. 6D-H). The average neurite length (Fig. 6D) increased more, relative to the Vehicle, for the younger male and female cohorts compared to the middle-aged female cohort. The middle-aged female cohort was the only cohort without a significant increase in average neurite length at any concentration, resulting in a significant difference in the relative increase of neurite length at 3400 nM RO48 between the younger cohorts. For the total neurite outgrowth (Fig. 6E), the middle-aged female cohort has a significant increase in the neurite outgrowth compared to Vehicle treatment and significantly larger relative change compared to the younger cohorts. The young adult male and female cohorts do not differ significantly, although, the young adult female cohort does have a significant increase in neurite length at the highest dose. All 3 cohorts significantly increased the number of valid neurons at doses ≥ 300 nM, although, the relative increase from the Vehicle treatment differs significantly between all 3 cohorts (Fig. 6F). The young adult female cohort is most responsive, followed by the young adult male and middle-aged female cohort. This data elucidates the sex and age-dependent effects of RO48 on adult neurons in vitro which are more profound at higher RO48 concentrations. More importantly, it demonstrates the need to test compounds in both sexes and age groups to determine demographic specific efficacies.
The age-dependent toxicity of 7-epi Paclitaxel. 7-epi Paclitaxel is an FDA-approved drug for use in patients with ovarian cancer 72 . 7-epi Paclitaxel stabilizes microtubule bundles, impairs organelle transport 73 , induces peripheral neuropathy through the CXCR1/2 pathway 74 , and reduces brain injury after repeated traumatic brain injuries in mice by inducing neurite growth and nerve regeneration 75 . Adult neurons isolated from the cortex of young adult male, young adult female, and middle-aged male mice were treated with 150 nM 7-epi Paclitaxel and Vehicle for 3DIV to analyze the average neurite length, total neurite outgrowth, and number of valid neurons (Fig. 7). 7-epi Paclitaxel significantly increases the average neurite length of all 3 age cohorts, yet only significantly decreases the total neurite outgrowth and number of valid neurons of the young adult cohorts without affecting the middle-aged male cohort. Therefore, screening 7-epi Paclitaxel in young adult neurons at 3DIV would yield an overall negative result while yielding an overall positive result in middle-aged male neurons suggesting a change in both the magnitude and direction of the effect. Studying the influence of 7-epi Paclitaxel in different demographics uncovers the need to conduct age-appropriate screenings to provide demographicspecific results and mitigate premature dismissal of potentially beneficial compounds.

Discussion
We developed a new screening platform utilizing primary adult mouse cortical neurons and demonstrated sex-and age-dependent effects of neuroactive compounds. This discrepancy between demographics supports the notion that future screenings must include both sexes and different age groups to account for sex-and age-dependent processes that may alter drug efficacy and even elicit opposite effects. Therefore, screening in multiple demographics is a necessary step in reducing unforeseen erroneous results and increasing confidence in the results. High content screenings are a vital process in drug discovery to rapidly identify potential candidates and pathways to achieve the desired therapeutic benefits. The use of cells comparable to the cellular targets in vivo is www.nature.com/scientificreports/ likely to increase the chance of success when moving a drug to pre-clinical and clinical settings. For this reason, cortical neuron screens are commonly used for finding novel therapeutics to increase axon regenerative capacity for neurotraumatic injuries 76,77 . Considering the many epigenetic 78,79 and metabolic 21 changes occurring in neurons as they age, the age-factor should be considered during screenings. For example, pro-inflammatory cytokines deemed harmful to adults are beneficial for the development of young neurons 25 and changes in mitochondrial functions impact SCI in an age-dependent manner 80 . Therefore, compounds that modulate cytokine production or mitochondrial functions may produce age-dependent effects 80 . 7-epi Paclitaxel is an example of such a phenomenon for the need to use demographic specific screens. Nonetheless, screening adult, even young adult, neurons has previously not been plausible. Additionally, neuronal cultures are usually grown using growth factors (BDNF, NGF, or NT3) 21,50,54 which are likely to conceal the beneficial effects of tested compounds. Thanks to the advancements made by Miltenyi in neuron dissociation and isolation, small volumes of primary adult neurons can be cultured 21,57 . We extensively modified this protocol to mass process larger volumes of cortical tissue with improved methods that reduce costs, increase processing speed, increase total number of cells per preparation, and increase yield and purity (based on qRT-PCR) to conduct screenings using neurons of all ages given enough brain tissue matter. The MetaXpress ® 6 automated analysis system from Molecular Devices provides an unbiased analysis at an unprecedented rate allowing for the high content morphology-based neuron screens. The analysis used herein is based on 3 parameters: average neurite length, total neurite outgrowth, and number of valid neurons. First, the software finds nuclei (DAPI) that are associated with neurites (TUBB3). Next, the software determines if the total associated nerites are ≥ 10 µm in length and only then considers the cell to be a valid neuron. This analysis method is biased against neurons of very little neurite growth but simultaneously protects the users from falsely accounting debris particles as valid neurons. Notably, using the valid neurons analysis does not allow for the determination of how many neurons survived or how many neurons initiated neurite growth, rather analyzes a combination of the two. Therefore, with this analysis method, we cannot directly conclude that increased valid neurons increases the survival rate, although is hypothesized to be highly associated with the average viability of the entire culture. Next, the software quantifies neurite outgrowth only from these valid neurons and divides that value from the number of valid neurons to determine the average total neurite outgrowth per neuron.
To determine the average neurite length, the total neurite outgrowth value is divided by the average number of branches found on only valid neurons. Therefore, the average neurite length analysis cannot determine if the neurites have equal sized branches or very long neurites with minuscule branching neurites. The software cannot specifically analyze the length of the longest neurite which is one factor in determining average neurite length. 7-epi Paclitaxel induces a noticeable increase in the length of the longest neurite (data not included) and significantly increases the average neurite length, therefore, the two analyses can be associated. More laborious analysis methods, such as neurite tracing 21 , can be used to further evaluate morphological changes. Regardless, our analyses provide enough information for determining which compounds have potential in preclinical settings and should be analyzed in further detail before further investment in preclinical trials.
Many variables went into consideration in the formation of the final neuron isolation, processing, and culturing protocol. Deciding the precise details or even inclusion of each step was determined by the effects of that step on each of the variables analyzed and the associated cost, labor, and time requirements. To assess the effects of compounds on neuron morphology, the neuron must be able to survive in culture which requires attaching to the surface and initiating neurite outgrowth and be healthy/viable enough to respond to environmental changes such as the addition of a drug. A protocol that maximizes neuron survival and viability is more versatile to other species, age groups, and regions of the CNS. Our protocol is tailored to cortical neurons, although, with slight modifications, can be used for neurons from other regions. We were able to culture young adult hippocampal and spinal neurons by adding the SM1 supplement in addition to B27 + and 10 ng/mL recombinant human brain derived neurotrophic factor (BDNF, Tonbo Biosciences, 21-8366-U010) to the neuron media. BDNF artificially added into culture was expected to increase the viability of neurons plated at low densities, although this was contrary to our findings. BDNF has been found to stop enhancing the survival of hippocampal neurons at ages above E17 81 even though tyrosine receptor kinase B (TrkB), the high-affinity receptor for BDNF, is present in the cortex of adult rodents 82 . It is plausible that B27 + contains factors that activate TrkB, therefore, the addition of more BDNF has residual effect. Increasing the concentration of debris removal solution from the recommended 22.5% to ≥ 25% reduces the number of valid neurons tremendously. Conversely, tissue samples containing large amounts of debris prevent the visualization and quantification of neurons and neurites. Although the removal of debris can enhance neuron survival and neurite outgrowth 83,84 , the debris removal solution may remove essential factors and biological agents that support survival and neuritogenesis. In fact, we observed improvements in survival when removing the debris removal step (not shown). However, this is accompanied with a lot of debris that make semi-automatic quantifications difficult. Since the purpose of the assay is to evaluate increases in neurite outgrowth, the protocol is designed to ensure minimal baseline increase in neurite outgrowth without affecting the number of valid neurons. To reduce potential off-target effects, cost, and time, the protocol minimizes reagent usage. Laminin coating was deemed unnecessary and excluded from the protocol (Fig. 1) and the lowest concentration of papain with maximal effect is recommended (Fig. 1). In terms of deciding between neural supplements, B27 + significantly improves all 3 variables compared to SM1 and NeuroBrew. The potential problem with using the best supplement such as B27 + is it may mask the true effects of compounds by providing an overabundance of growth factors and nutrients. This theory was disproven by the significant effects seen with different drug treatments in culture supplemented with B27 + (Figs. 6, 7). Increasing the number of neurons through proper media supplementation allows for larger screens. B27 + provides a nice compromise between survival and neurite growth, with a well characterized fetal bovine serum (FBS) free composition, allowing for accurate assessment of the effects of compounds on adult neurons. www.nature.com/scientificreports/ The cell plating density is an important aspect of cell cultures 85 and can dictate the survival of neurons 52,86 . For hippocampal neurons, cell plating density influences synapse formation 87 , maturation, and intensity of electrical activity 88 . Our data demonstrates the effects of plating density on viability and morphology. At densities < 5000 cells/well, the neurons are less likely to produce the typical elongated neurites characteristic of a healthy neuron and typically too few neurons to accurately determining differences between treatment groups; variability between treatment groups is inversely proportional to the number of neurons quantified. Adult neurons plated at ≥ 15,000 cells/well have extended and overlapping neurite outgrowth that makes accurate quantification difficult. Having a plating density that is too high (≥ 15,000 cells/well) also leads to an overpopulated culture that grows at an expedited rate which can mask the effects of compounds. Therefore, subsequent experiments and the preferred plating density is 10,000 cells for a well with an area of 0.056 cm 2 . As we increased plating density, we observed an exponential increase in number of valid neurons and there is a significant correlation between neurite outgrowth and plating density. Therefore, extrinsic factors associated with plating density may influence both the number of valid neurons and neurite morphology. One potential factor is glutamine released by neighboring neurons 89 . Glutamine is artificially added to the culture media at a final concentration of 2 mM to increase neuron viability yet may not be enough or may only be one of the factors associated with both viability and plating density. Our improved protocol increases the neuron enrichment, as shown by qRT-PCR. However, other neural cells in culture, even if in low abundance, may also influence the density associated effects. Astrocytes release neurotrophic factors, such as BDNF, that augment neuron survival and function 90 . Endothelial cells may also be present in our culture, as we do not remove them with our protocol. These cells can secrete several factors, including GDNF, that can increase neuron survival and neurite growth 91,92 . While further elucidation is required to determine which organic molecules are released and by which cells, our data identified an appropriate cell density that allows to screen for compounds modulating neuron survival and neurite growth.
Laminin is a glycoprotein that is part of the extracellular matrix and is involved in cell differentiation, attachment, and growth 93 , partially through its interaction with integrins 94 . Laminin is commonly used as a precoating substrate to assist in the attachment of neurons and can increase the survival and growth of human pluripotent stem cell-derived neurons 95 . The addition of an extra layer of laminin coating on top of the PDL substrate further enhances attachment, survival, and growth of neural precursor cells 96 . Therefore, the addition of a laminin coating was tested on adult neurons with and without B27 + to assess its effects on cell attachment and growth. To our surprise, laminin did not impact neurite morphology, or the number of valid neurons and effects were not masked by B27 + supplementation. Therefore, the isolated adult cortical neurons do not seem receptive to laminin. We specifically used mouse derived laminin for this experiment to reduce the potential for inflammatory reactions and nonhomology between other species. We hypothesize that adult neurons have a reduced number of integrin receptors, or that downstream pathways are activated to a lesser extent relative to younger neurons resulting in reduced response to laminin coatings. Dorsal root ganglia neurons from P0 rats are able to increase integrin expression in culture while adult neurons are uncapable of this adaptation resulting in reduced survival and neurite outgrowth 97 . Transgenic expression of integrins in adult neurons restores their neurite regenerating capacity 98 . Therefore, integrin-dependent substrates, such as laminin, are not necessary to culture adult neurons for short period of time. This observation is in fact of high interest, as it reduces both the cost and time of screening, while minimizing the number of confounding variables. Indeed, not using laminin coatings will allow for screening molecules activating the downstream molecular signaling pathways without potential confounding factors.
The dissociation of neurons from the extracellular matrix is an essential part of neuron isolation. Traditional methods include incubating the brain tissue in digestive enzymes 99 , digesting through mechanical means 100 , or both 101 . To maximize neuronal yield, we opted for using both methods. Papain, which has been used in previous studies to dissociate neurons 102 , produced the highest number of valid neurons when used at concentrations ≥ 0.3 mg/mL. It is unknown if papain increases the enzymatic dissociation of neurons from the extracellular matrix or if it is gentler on adult neurons resulting in higher rates of survival compared to other dissociation enzymes. Different incubation times, temperatures, and mechanical dissociation methods were also tested to maximize the number of valid neurons. Lower temperatures and incubation times from Miltenyi's original protocol 59 were assessed to determine if milder conditions would increase neuron viability and survival. With the use of papain specifically, the reduction in temperature and time yielded significantly less valid neurons hypothesized to be from reduced enzyme efficiency as it is unconceivable how decreasing the temperature and incubation period would reduce the viability of neurons. A very large improvement to traditional methods was achieved with the gentleMACS™ Octo Dissociator with Heaters even when comparing to other methods of agitation. When no agitation method was used, the number of valid neurons were further reduced (data not included). It is unknown why the agitation induced by the gentleMACS™ Octo Dissociator with Heaters cannot be easily replicated using other methods. It may be providing the most effective amount of agitation while other methods may be over or under agitating the brain tissue. Some dissociation protocols require trituration, which are prone to bring great batch to batch variability 103 . Our protocol aims to maximize rigor and reducibility to be more sensitive to effect sizes. Through extensive testing, Miltenyi's original dissociation method is the best tested method to increase the number of valid adult neurons. Figure 1 demonstrates the effects the dissociation enzymes have on the viability and growth capacity of neurons. The images also demonstrate that papain increases neuronal purity. RT-qPCR based RNA expression analysis confirmed the modified protocol (using papain instead of P&A) has higher neuronal yield and purity relative to Miltenyi's original protocol which concurs with the images; automated morphology analysis only confirmed greater number of valid neurons as the other variables were not analyzed (Fig. 1A-C). However, we did not conduct RNA expression analysis directly comparing the effects of substituting P&A with papain on neuronal yield and purity. Further experimentation is required to elucidate the effects of various dissociation enzymes on neuron health. www.nature.com/scientificreports/ Many potent compounds are hydrophobic and require the use of special solvents, which can be harmful to neurons. DMSO is a class 3 solvent (Food and Drug Administration) used to increase the solubility of hydrophobic compounds 67 . Therefore, many high content screens utilize DMSO to dissolve and prevent precipitation of hydrophobic compounds in cell culture media 68,69 . The issue of using DMSO as a vehicle is its toxicity to neurons and can alter their properties. A brief treatment of 0.05% DMSO induces neurophysiological changes to both hippocampal and cortical neurons 104 . DMSO concentrations ≥ 0.5% induce neurite retraction of primary embryonic neurons 105 . The effects of DMSO on adult cortical neurons have not need assessed before, and it is necessary to determine the best concentration minimizing the impact of DMSO on neurons while being high enough to allow the solubilization of most compounds tested in future screens. Based on our findings, it is recommended that the use of DMSO in high content screenings in adult neurons follow 2 criteria: (1) The use of DMSO is minimized and is ≤ 0.05% of the culture media; (2) the concentration of DMSO is kept consistent between all cohorts to reduce the confounding effect of DMSO on the analysis. The mechanism of such outcomes has yet to be elucidated. One possibility is the reduction of ERK phosphorylation. Indeed, phosphorylation of ERK, induced by alpha-lipoic acid in mouse neuroblastoma N2a cells, promotes neurite outgrowth 106 , while DMSO has been shown to reduce ERK phosphorylation in blood cells 107 . Therefore, DMSO may be reducing the neurite outgrowth potential of neurons by modulating the ERK pathway. DMSO also reduces the number of valid neurons (total neurite outgrowth of ≥ 10 µm) in a dose-dependent manner which is associated with a reduction in neurite outgrowth (Fig. 5). Therefore, it is plausible the reduction in neurite outgrowth is merely from the reduction in neuron survival. The sensitivity of adult neurons to DMSO results in screening limitations, therefore, other solvents should be considered for testing compounds at higher concentrations.
Due to the vast range of responses the general population has towards a given therapeutic 108,109 , it is vital to consider the age and sex factor in the development of new therapeutics. The cells isolated from that demographic are expected to respond differently as well. Here, we determined the demographic specific effects of RO48 and 7-epi Paclitaxel in our targeted screen to demonstrate the necessity for conducting demographic specific screens in neurons to ensure the compounds influence the target audience. Our in vitro cell cultures have identical conditions between age groups and are from very genetically similar mice. Thus, cell cultures allow us to determine how the neuron's sex chromosomes (sex difference) or epigenetic profile (age-dependent effect) impact neuronal phenotype without known confounding variables. Through our specific processing and culturing methodology, we discovered both sex and age differences in response to exposure to different drugs.
We demonstrated that RO48 induces both sex-and age-dependent effects on neuron morphology and viability. RO48 activates mTORC1/2 and PI3K and decreases the phosphorylation of S6-926 70 and S6K1 which are negative regulators of neurite growth 110 . Overall, RO48 present positive effects regardless of sex and age. RO48 significantly increases the average of neurite length in young adult neurons and increases the total neurite outgrowth only in older females. RO48 is very effective at increasing the number of valid neurons, which is expected since both mTOR and S6K1 increase neuron survival 111 . Intriguingly, the potency of RO48 to increase the number of valid neurons differs significantly with age and sex. One explanation for the smaller effect of RO48 at increasing the number of valid neurons with age is the age-dependent reduction in mitochondrial function. Indeed, S6K1 enhances mitochondrial ATP production 112 which is important for axon growth and cell survival and could be a mechanism of action of RO48. However, we recently demonstrated that aging reduces neuronal mitochondrial capacity and efficiency 21 . Therefore, it is possible that the increase in ATP production in presence of RO48 is not as potent in older neurons, leading to a reduced activity of RO48. PI3K may also be involved in the age-dependent effects. PI3K decreases as cells age 113 . PI3K reduces neurite branch formation 114 which would lead to an increase in the average neurite length. Since only the younger cohorts have increased neurite length in response to RO48, there may be an age-dependent response to PI3K modulation or age-dependent ability to express PI3K resulting in the significant differences in average neurite length in response to RO48. The single instance RO48 was not beneficial was in the average neurite length on middle-aged female neurons. There is a decrease in the ratio of phosphorylated mTOR in middle-aged naked mole rats compared to their young adult counterparts 115 which is part of the mTOR activation process 116 . The mTOR pathway is an essential part of neurite formation [117][118][119] . Since there was a significantly greater increase in total neurite outgrowth in the older cohort in response to RO48 relative to their younger counterparts, and a greater increase in average neurite length in younger cohorts relative to the middle-aged female cohort, it may be plausible that mTOR phosphorylation induces neurite growth in all cohorts in a similar manner. The differences between age groups may be due to the management of neurite elongation. Indeed, we have shown at 7DIV, neurons from aged mice have more branching 21 . This may be due to neurons from younger mice better consolidating and focusing neurite growth into few branches for the objective of producing axons. Nonetheless, direct experimentation is required to elucidate the pathways responsible these differences.
The sex differences of RO48 might be due to the activation of sex-dependent pathways. PI3K inhibition increases hepatic GSH content, antioxidant genes, and catalase in males but not in females 120 . The increase of catalase activity is correlated with reduction in neurite outgrowth 121 , therefore, one intriguing possibility is that the influence of PI3K on neurite outgrowth is sex-dependent. Another possibility is the S6 phosphorylation rate difference. Indeed, female mice have significantly higher rates of S6 phosphorylation in both the liver and heart 122 , and therefore, may be more susceptible to the reduction in S6 phosphorylation induced by RO48 70 . This remains to be determined in cortical neurons. Inhibition of mTOR only increases brain proteasome activity in females 123 . Proteasome inhibition is a potential therapeutic option for increasing neurite outgrowth 124 , therefore, sex differences in neurite outgrowth through the mTOR-proteasome pathway would not be surprising. 7-epi Paclitaxel is an FDA-approved drug for use in patients with ovarian cancer 72 . 7-epi Paclitaxel stabilizes microtubule bundles, impairs organelle transport 73  www.nature.com/scientificreports/ neurotoxicity occurs sooner and more frequently in older metastatic breast cancer patients treated with 7-epi Paclitaxel compared to younger patients 125 . This discrepancy may be due to older patients generally being more vulnerable to neurotoxicity regardless of the treatment 126,127 . Conceivably, the increased 7-epi Paclitaxel-induced toxicity in older patients may be due to pharmacokinetic changes in aged patients that lead to prolonged drug activity and toxicity and increased drug sensitivity 128 . Therefore, it is plausible 7-epi Paclitaxel is less toxic to older neurons yet still more toxic to older patients. Interestingly, the CXCR1/2 expression is increased with age in rat cortical neurons 129 . The lower baseline of CXCR1/2 levels in younger neurons may explain the increased neurotoxicity relative to middle-aged neurons. Further direct experimentation is required to elucidate the agedependent neurotoxicity of 7-epi Paclitaxel.

Conclusion
Screening for compounds directly in adult cortical neurons has been so far unrealistic. Here, we established a new protocol that augments our ability to screen directly in primary mouse adult cortical neurons (Fig. 8). We conducted targeted screenings and demonstrated both age-and sex-dependent effects of multiple compounds. We established that: (1) dosage can intrinsically be sex-dependent; (2) screening in younger demographics will cause premature dismissal of compounds beneficial to older demographics; and (3) aging alters neuronal characteristics and therefore, must be considered in future screenings, especially for age-associated neurological disorders. This novel methodology is expected to strengthen the drug discovery process for neurological disorders and neurotraumatic injuries by providing more relevant in vitro data increasing the likelihood of preclinical and clinical success. Plus Supplement (ThermoFisher Scientific, A3582801). We followed the respective instruction manuals for all neuronal supplement use (B27 + , SM1, NeuroBrew). All supplements came in a 50× solution and were diluted to a final concentration of 1× following manufacturer's instructions. Unless noted otherwise, cells were added onto PDL coated wells and placed inside a 5% CO 2 incubator set at 37 °C for the stated days in vitro (DIV). Unless noted otherwise, 10,000 cells were plated per well with 0.056 cm 2 growth area. The neuron isolation, processing, and plating procedure described herein is referred to as the 'Modified Protocol' .
RT-qPCR for determining cell culture purity. RT-qPCR assay was replicated as previously described 21 .
Briefly, the cells isolated from cortical tissue of young adult male mice were pelleted after the completion of the respective protocol followed by the extraction of RNA immediately after. Directzol RNA micro-prep columns (Zymo, R2061) are used to extract RNA from neurons directly following neuron isolation. RNA concentration was measured using the Thermo Scientific™ NanoDrop 2000. Quantabio cDNA Synthesis kit (Quanta, 95047) was used to synthesize cDNA before conducting qPCR using the Quantabio PerfeCTa ® SYBR ® Green FastMix ® (Quanta, 95073) on the ViiA7 Real Time PCR system (Life Technologies). The neuron enrichment in the negative fraction was calculated as previously described 21 , − ΔCT of ΔNeuN against ΔGFAP and ΔGLAST using the formula: − ΔCT = − (ΔCT NeuN − (SQRT(ΔCT GFAP 2 + ΔCT GLAST 2 ))). CT was calculated for each group based on the absolute CT per primer subtracted by the respective CT of the negative control to reduce background noise. For each group, RNA was extracted from 3 separate isolation procedures from young adult males and each sample was analyzed in triplicate. β-actin was used as the internal housekeeping gene control to normalize gene expression. No outliers were detected nor omitted. Primers used to identify the main cellular constituents Immunocytochemistry. Cell cultures were fixed with 4% paraformaldehyde (PFA, 15 min) after the completion of the respective experiment. After fixation, immunocytochemistry was conducted by first washing the cells with DPBS 3 times, then incubating in 5% normal horse serum for 60 min to block nonspecific binding (VWR, 102643-676). Afterwards, the cells were incubated in 1:500 TUBB3 (BioLegend, 801202) for 16 h. followed by another 3 washes with DPBS and incubation in 1:500 Alexa Flour 488 (ThermoFisher Scientific, A32723) and 1:10,000 DAPI (VWR, 95059-474) for 60 min, all conducted at room temperature. After the completion of immunocytochemistry, the cells were preserved in Fluoromount-G Mounting Medium (Ther-moFisher Scientific, 00-4958-02) until imaging. All experimentation after incubation in secondary antibody was performed in the absence of light.
Analysis of neurite outgrowth and survival. Representative images in figures were imaged using 20×/63× objectives on a Zeiss Axio Observer system. For automated image acquisition, the × 20 magnification lens of the ImageXpress (IXM) Micro Confocal High-Content Imaging System (Molecular Devices, San Jose, CA) was used along with the Neurite Outgrowth Analysis Module in MetaXpress ® 6 software (Molecular Devices) for automated image analysis, a system previously used to image and analyze changes in neuron morphology 28,[130][131][132] . Using the × 20 magnification, 16 separate images (with 10% overlap) were required to sustain > 90% coverage of each well while avoiding the walls. Three variables were quantified.
1. Valid neurons Total number of cells in a well that are both DAPI and TUBB3 positive and with total neurite outgrowth of ≥ 10 µm. 2. Total neurite outgrowth Sum of the lengths of all the neurites from a valid neuron. This is then averaged over all the valid neurons in a well. 3. Average neurite length The total length of all the neurites from a valid neuron divided by the number of neurites and branches of that cell. This is then averaged over all the valid neurons in the well.

Data analysis.
Normally distributed data was analyzed using unpaired t-test when comparing 2 means and one-way analysis of variance (ANOVA) with Tukey's or Dunnett's post-hoc test when comparing > 2 means. Tukey's post-hoc test is used to compare > 2 means all with one another. Dunnett's post-hoc test is used to compare ≥ 2 means to the mean of a particular group. Two-way ANOVA was used to analyze the effects of 2 independent variables on the expected outcome. Tukey's multiple comparisons test was used to compare the means of each cohort with the means of all other cohorts. Dunnett's multiple comparisons test was used to compare the means of each cohort to the mean of a particular cohort. Šídák''s multiple comparisons test was used to compare the means of 2 independent groups for all cohorts. Simple liner regression was conducted to determine goodness of fit (R 2 ) and to determine the correlation between 2 independent variables. Linear regression t-test was used to compare the slopes of 2 independent regression lines. Analysis and graphing were conducted using Graphpad Prism 9. All graphs represent the data mean with error bars illustrating the standard error of the mean (SEM).
Compounds. RO48 (generously provided by Drs. Lemmon, Bixby, and Al-Ali from Miami Project to Cure Paralysis, University of Miami), (S)-H-1152 (Cayman Chemical Company, 10007653), and 7-epi Paclitaxel (Cayman Chemical Company, 20741). All compounds are added to the media at the time of cell plating. No media changes occur during the entire duration of the assay; therefore, the cells are incubated with the compounds for the entire duration of the assay.
Institutional review board statement. The study was conducted and approved by the Institutional Animal Care Committee at Texas A&M University (IACUC 2018-0048). All the experiments reported here were reviewed and approved by the and were consistent with the ARRIVE guidelines for animal care and use. All methods were carried out in accordance with the guidelines and regulations from the Institutional Biosafety Committee at Texas A&M (IBC-2018-011). www.nature.com/scientificreports/